1. Field of the Invention
The present invention provides a method for the determination of the presence of an analyte and an analytical instrument capable of performing the method.
2. Description of Related Art
The invention is useful in the field of analytics or diagnostics, particularly in the diagnostics of nucleic acids. The analysis of nucleic acids has been improved considerably by the invention of the Polymerase Chain Reaction (PCR) as disclosed in EP 0 200 362 and EP 0 201 184. During the course of this method, the amount of nucleic acids is increased at least partially exponentially, as theoretically from each nucleic sequence present in the reaction mixture in each reaction cycle an additional nucleic acid is created, and each of the nucleic acids can act as a template for the creation of a further nucleic acid sequence in the following reaction cycle. The amount of nucleic acids created is limited by e.g., the amount of reagents, like enzymes, primers and nucleotides, contained in the reaction mixture. Therefore, the concentration plotted versus the time or cycle number of the PCR resembles an (asymmetric) sigmoid curve.
A further improvement of PCR is the so-called Real-Time-PCR. In this method, a signal is created and detected during amplification. The signal is representative of the amount of nucleic acids created during amplification and thus present in the reaction mixture. In a first embodiment, e.g. disclosed in EP 0 512 334, the signal is created by a compound capable of intercalating into double stranded nucleic acids while changing its fluorescence properties. In another embodiment, as disclosed in EP 0 543 942, each extension reaction of a primer leads to the cleavage of a probe, labeled by a quencher and an emitter dye such that when cleaving the probe, the quencher cannot quench the light emission of the reporter dye, so that a signal can be detected.
The determination of the amount of nucleic acid originally present in the sample prior to amplification (quantification or quantitation) has been the goal of several investigations. Generally, the higher the amount the smaller the number of reaction cycles needed to receive a defined intensity of the signal (threshold). The earliest calculations therefore were based on the determination of the threshold cycle (CT)-value. The higher the CT-value, the lower the original amount of nucleic acid present. Obviously, the (integer) number of reaction cycles conducted can only be a very rough estimate of the amount originally present. Thus, in a further attempt to determine concentrations the signal intensities lying between distinct measurement data were interpolated (linear or logarithmically). These interpolation based methods have some deficiencies, e.g., they are sensitive to the presence of imprecise signal measurements or measurement outliers (e.g., spikes). To avoid this, algorithms have been established to create continuous growth curves from a defined number of measurements during the amplification reaction. One example of such an algorithm is the so-called Sawitzky Golay Filter. In EP 0 686 699 there is described a conditional recursive formula which can be used for fitting of measured data to a theoretical curve. However, the application is cumbersome and the fitting process is not described. The algorithm lead to strong parameter correlations and inaccurate results in certain cases.
In WO 97/46714 methods of monitoring hybridization after polymerase chain reaction (PCR) are disclosed. In particular, the application discloses that the sensitivity of an initial template quantification with fluorescence vs. cycle number plots can be increased by analysis of product melting curves to control for nonspecific amplification and by non-linear regression fitting Levenberg-Marquard curve-fitting algorithms.
In Biotechnology Letters 24, 2002, 2053-2056 there is disclosed a method to determine the amplification efficiency of RT-PCR using a four parametric sigmoid model.
In Biochemical and Biophysical Research Communications 294, 2002, 347-353 there is also described a PCR simulation method for determining the efficiency of PCR.
These two mathematically equivalent four parameter models provide limited accuracy especially in the areas which usually are critical for an exact result calculation. The baseline is forced to be constant and the simple sigmoid term is not capable of approximating the full complexity of a general growth curve. Therefore, the exponential phase is approximated with limited accuracy as can be visually observed in the graphs of the two papers. This leads to a result with limited accuracy, e.g. CT.
It was the object of the present invention to improve the quantitative analysis, particularly to provide a fully automatic method using a mathematical calculation to better estimate the whole growth curve, especially to correct measurement imprecision and possible measurement spikes.